Seungeun Oh

Multiple Phases of Chondrocyte Enlargement Underlie Differences in Skeletal Proportions

The wide diversity of skeletal proportions in mammals is evident upon a survey of any natural history museums collections and allows us to distinguish between species even when reduced to their calcified components. Similarly, each individual is comprised of a variety of bones of differing lengths. The largest contribution to the lengthening of a skeletal element, and to the differential elongation of elements, comes from a dramatic increase in the volume of hypertrophic chondrocytes in the growth plate as they undergo terminal differentiation. However, the mechanisms of chondrocyte volume enlargement have remained a mystery. Here we use quantitative phase microscopy to show that mammalian chondrocytes undergo three distinct phases of volume increase, including a phase of massive cell swelling in which the cellular dry mass is significantly diluted. In light of the tight fluid regulatory mechanisms known to control volume in many cell types, this is a remarkable mechanism for increasing cell size and regulating growth rate. It is, however, the duration of the final phase of volume enlargement by proportional dry mass increase at low density that varies most between rapidly and slowly elongating growth plates. Moreover, we find that this third phase is locally regulated through a mechanism dependent on insulin-like growth factor. This study provides a framework for understanding how skeletal size is regulated and for exploring how cells sense, modify and establish a volume set point.

Dynamics extracted from fixed cells reveal feedback linking cell growth to cell cycle

Biologists have long been concerned about what constrains variation in cell size, but progress in this field has been slow and stymied by experimental limitations. Here we describe a new method, ergodic rate analysis (ERA), that uses single-cell measurements of fixed steady-state populations to accurately infer the rates of molecular events, including rates of cell growth. ERA exploits the fact that the number of cells in a particular state is related to the average transit time through that state. With this method, it is possible to calculate full time trajectories of any feature that can be labelled in fixed cells, for example levels of phosphoproteins or total cellular mass. Using ERA we find evidence for a size-discriminatory process at the G1/S transition that acts to decrease cell-to-cell size variation.

Imaging voltage-dependent cell motions with heterodyne Mach-Zehnder phase microscopy

We describe a heterodyne Mach-Zehnder interferometric microscope capable of quantitative phase imaging of biological samples with subnanometer sensitivity and frame rates up to 10 kHz. We use the microscope to image cultured neurons and measure nanometer-scale voltage-dependent motions in cells expressing the membrane motor protein prestin.

Quantitative DIC microscopy using an off-axis self-interference approach.

Traditional Normarski differential interference contrast (DIC) microscopy is a very powerful method for imaging nonstained biological samples. However, one of its major limitations is the nonquantitative nature of the imaging. To overcome this problem, we developed a quantitative DIC microscopy method based on off-axis sample self-interference. The digital holography algorithm is applied to obtain quantitative phase gradients in orthogonal directions, which leads to a quantitative phase image through a spiral integration of the phase gradients. This method is practically simple to implement on any standard microscope without stringent requirements on polarization optics. Optical sectioning can be obtained through enlarged illumination NA.

Size homeostasis in adherent cells studied by synthetic phase microscopy

Significance Accurate measurement of cell size is critical in studies of cell growth. Optical methods based on interferometry are known to be suitable for attached cells, but the existing techniques were originally designed for thin samples and are not ideal for thick ones, such as mitotic cells. Synthetic phase microscopy (SPM), a new tomographic interferometric method, offers an elegant solution to this problem. This paper demonstrates the ability of SPM to measure the growth of mammalian cells accurately, and it demonstrates a clear requirement for feedback in the growth process. The coupling of the rate of cell growth to the rate of cell division determines cell size, a defining characteristic that is central to cell function and, ultimately, to tissue architecture. The physiology of size homeostasis has fascinated generations of biologists, but the mechanism, challenged by experimental limitations, remains largely unknown. In this paper, we propose a unique optical method that can measure the dry mass of thick live cells as accurately as that for thin cells with high computational efficiency. With this technique, we quantify, with unprecedented accuracy, the asymmetry of division in lymphoblasts and epithelial cells. We can then use the Collins–Richmond model of conservation to compute the relationship between growth rate and cell mass. In attached epithelial cells, we find that due to the asymmetry in cell division and size-dependent growth rate, there is active regulation of cell size. Thus, like nonadherent cells, size homeostasis requires feedback control.

Resonant microchannel volume and mass measurements show that suspended cells swell during mitosis

Suspended cells transiently increase their volume during mitosis because of ion exchange through the plasma membrane.

Improved phase sensitivity in spectral domain phase microscopy using line-field illumination and self phase-referencing

We report a quantitative phase microscope based on spectral domain optical coherence tomography and line-field illumination. The line illumination allows self phase-referencing method to reject common-mode phase noise. The quantitative phase microscope also features a separate reference arm, permitting the use of high numerical aperture (NA > 1) microscope objectives for high resolution phase measurement at multiple points along the line of illumination. We demonstrate that the path-length sensitivity of the instrument can be as good as 41 pm/square root of Hz, which makes it suitable for nanometer scale study of cell motility. We present the detection of natural motions of cell surface and two-dimensional surface profiling of a HeLa cell.

Ultraviolet refractometry using field-based light scattering spectroscopy

Accurate refractive index measurement in the deep ultraviolet (UV) range is important for the separate quantification of biomolecules such as proteins and DNA in biology. This task is demanding and has not been fully exploited so far. Here we report a new method of measuring refractive index using field-based light scattering spectroscopy, which is applicable to any wavelength range and suitable for both solutions and homogenous objects with well-defined shape such as microspheres. The angular scattering distribution of single microspheres immersed in homogeneous media is measured over the wavelength range 260 to 315 nm using quantitative phase microscopy. By least square fitting the observed scattering distribution with Mie scattering theory, the refractive index of either the sphere or the immersion medium can be determined provided that one is known a priori. Using this method, we have measured the refractive index dispersion of SiO(2) spheres and bovine serum albumin (BSA) solutions in the deep UV region. Specific refractive index increments of BSA are also extracted. Typical accuracy of the present refractive index technique is <or=0.003. The precision of refractive index measurements is <or=0.002 and that of specific refractive index increment determination is <or=0.01 mL/g.

Differential Heterodyne Mach-Zehnder Interferometer for Measurement of Nanometer-Scale Motions in Living Cells

We have developed a novel differential heterodyne Mach-Zehnder interferometer for measuring sub-nanometer displacements in single cells. We describe efforts to measure voltage-dependent refractive index changes and mechanical fluctuations in cultured single neurons.